泡桐丛枝植原体tRNA异戊烯基焦磷酸转移酶基因克隆、原核表达及功能分析

【目的】为了鉴定植原体tRNA异戊烯基焦磷酸转移酶基因(tRNA-ipt)的表达及蛋白功能,探索植原体致病机理。【方法】对泡桐丛枝、桑萎缩、长春花绿变及苦楝丛枝植原体tRNA-ipt基因完整序列进行PCR扩增和生物信息学分析。对泡桐丛枝植原体tRNA-ipt基因进行原核表达并制备抗体。利用Western blot和FITC间接免疫荧光显微镜检测其在植原体中的表达。使用分光光度计分析该基因对大肠杆菌生长的影响,用ELISA测定转化菌株细胞分裂素含量。【结果】首次发现泡桐丛枝、桑萎缩、长春花绿变及苦楝丛枝植原体中完整tRNA-ipt基因,大小为876 bp,编码291个氨基酸,且N端均含有ATP/GTP结合位点保守序列(GPTASGKT)。4种植原体tRNA-IPT之间的氨基酸序列相似率为99.1%-99.5%,与同组植原体同源性在95.4%-99.3%,与其他组植原体同源性低于70%。SDS-PAGE结果显示tRNA-IPT蛋白在大肠杆菌中得到表达。首次获得泡桐丛枝植原体tRNA-IPT抗体并检测到该蛋白在泡桐发病组织中的特异表达。经过对转化菌株生长曲线及玉米素含量的测定,发现该基因能促进大肠杆菌后期生长和玉米素核苷的积累。【结论】4种植原体tRNA-ipt基因编码相同特性的功能蛋白,泡桐丛枝植原体tRNA-IPT蛋白能够在植原体中表达,根据该基因对异源菌株生长速率和激素合成的影响推断该蛋白可能参与植原体的细胞分裂素合成,在致病过程中起到重要作用。     

两株淡水微囊藻的藻蓝蛋白基因间隔序列(PC-IGS)分析

对2株编号为003和004的淡水水华微囊藻(Microcystis.sp)的藻蓝蛋白基因间隔序列进行测定,获得长度均为608bp的2条序列。同时从GenBank中获取铜绿微囊藻(Microcystis aeruginosa Kütz,NCBI序列号AJ003179)及惠氏微囊藻(Microcystis wesenbergii,NCBI序列号AF385391)的序列。分别运用MEGA3及ClustalX(Version1.83)软件对这4株藻的PC-IGS序列进行碱基组成分析和序列比对。碱基组成的比对结果表明4株藻的G+C含量分别为003(50.5%),004(51.7%),铜绿微囊藻(50.7%),惠氏微囊藻(52.3%),相差范围在0.2%~1.8%之间,其结果不足以区分这四株微囊藻;序列比对则表明003号藻株与铜绿微囊藻和惠氏微囊藻的序列相似性分别为100%和88.35%,而004号藻株与铜绿微囊藻和惠氏微囊藻的序列相似性比较结果为95.13%和89.04%。此外,文章还探讨了PC-IGS序列作为微囊藻种间鉴定分子标记的可行性。     

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.     

Nitrogen deposition enhances moss growth,but leads to an overall decline in habitat condition of mountain moss‐sedge heath

Ecosystems are subject to multiple, natural and anthropogenic environmental influences, including nitrogen (N) deposition, land use and climate. Assessment of the relative importance of these influences on biodiversity and ecosystem functioning is crucial for guiding policy and management decisions to mitigate global change; yet, few studies consider multiple drivers. In the UK, ongoing loss of the internationally important arctic/alpine moss‐sedge community, Racomitrium heath, has been linked to elevated N deposition, high grazing pressures and their combination; however, the relative importance of these drivers remains unclear. We used environmental gradients across the habitat's European distribution (UK, Faroes, Norway and Iceland) to investigate the relative impact of N deposition and grazing pressure, as well as climate, on the condition of the dominant moss species, Racomitrium lanuginosum. Key variables including tissue chemistry, growth and cover were measured at 36 sites, and multiple linear regressions were used to examine the relative importance of the drivers across sites. Our results clearly show that regional variation in the condition of R. lanuginosum across Europe is primarily associated with the impacts of N deposition, with climate (air temperature) and grazing pressure playing secondary roles. In contrast to previous experimental studies, we found moss growth to be stimulated by elevated N deposition; this apparent discrepancy may result from the use of artificially high N concentrations in many experiments. Despite increased growth rates, we found that moss mat depth and cover declined in response to N deposition. Our results suggest that this is due to increased decomposition of material in the moss mat, which ultimately leads to loss of moss cover and habitat degradation. This study clearly demonstrates both the key role of N deposition in degradation of Racomitrium heath and the importance of observational studies along natural gradients for testing predictions from experimental studies in the real world.     

A maize ADP‐ribosylation factor ZmArf2 increases organ and seed size by promoting cell expansion in Arabidopsis

ADP‐ribosylation factors (ARFs) are small GTP‐binding proteins that regulate a wide variety of cell functions. Previously, we isolated a new ARF, ZmArf2, from maize (Zea mays). Sequence and expression characteristics indicated that ZmArf2 might play a critical role in the early stages of endosperm development. In this study, we investigated ZmArf2 function by analysis of its GTP‐binding activity and subcellular localization. We also over‐expressed ZmArf2 in Arabidopsis and measured organ and cell size and counted cell numbers. The expression levels of five organ size‐associated genes were also determined in 35S::ZmArf2 transgenic and wild‐type plants. Results showed that the recombinant ZmArf2 protein purified from Escherichia coli exhibited GTP‐binding activity. Subcellular localization revealed that ZmArf2 was localized in the cytoplasm and plasma membrane. ZmArf2 over‐expression in Arabidopsis showed that 35S::ZmArf2 transgenic plants were taller and had larger leaves and seeds compared to wild‐type plants, which resulted from cell expansions, not an increase in cell numbers. In addition, three cell expansion‐related genes, AtEXP3, AtEXP5 and AtEXP10, were upregulated in 35S::ZmArf2 transgenic lines, while the expression levels of AtGIF1 and AtGRF5, were unchanged. Collectively, our studies suggest that ZmArf2 has an active GTP‐binding function, and plays a crucial role in growth and development in Arabidopsis through cell expansion mediated by cell expansion genes.     

Construction of a recombinant BHV-1 expressing the VP1 gene of foot and mouth disease virus and its immunogenicity in a rabbit model

Foot-and-mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle. Using bovine herpesvirus type 1 (BHV-1) as a gene delivery vector for development of live-viral vaccines has gained widespread interest. In this study, a recombinant BHV-1 was constructed by inserting the synthetic FMDV (O/China/99) VP1 gene in the the gE locus of BHV-1 genome under the control of immediately early gene promoter of human cytomegalovirus (phIE CMV) and bovine growth hormone polyadenylation (BGH polyA) signal. After homologous recombination and plaque purification, a recombinant virus named BHV-1/gE⁻/VP1 was acquired and identified. The immunogenicity was confirmed in a rabbit model by virus neutralization test and enzyme-linked immunosorbent assay (ELISA). The result indicated that the BHV-1/gE⁻/VP1 has the potential for being developed as a bivalent vaccine for FMD and IBR.     

两栖类皮肤抗菌多肽及其应用

抗菌多肽广泛分布于动物、植物,用于抵御细菌、真菌、病毒和原虫,在进化上是一类非常古老而有效的天然防御物质。两栖动物的后天获得性免疫系统与哺乳动物相比极为脆弱,它们在长期的进化历程中演化形成了一套非常有效的抵御微生物侵袭的防御系统,这套系统主要就是其裸露皮肤表面的抗菌多肽(又称初级免疫或者先天免疫系统)。本文结合本实验室近年的研究工作,对两栖类皮肤抗菌多肽的结构、功能及应用作一简要综述。     

Replication Study in a Japanese Population to Evaluate the Association between 10 SNP Loci,Identified in European Genome-Wide Association Studies,and Type 2 Diabetes

AimWe performed a replication study in a Japanese population to evaluate the association between type 2 diabetes and 7 susceptibility loci originally identified by European genome-wide association study (GWAS) in 2012: ZMIZ1, KLHDC5, TLE1, ANKRD55, CILP2, MC4R, and BCAR1. We also examined the association of 3 additional loci: CCND2 and GIPR, identified in sex-differentiated analyses, and LAMA1, which was shown to be associated with non-obese European type 2 diabetes.MethodsWe genotyped 6,972 Japanese participants (4,280 type 2 diabetes patients and 2,692 controls) for each of the 10 single nucleotide polymorphisms (SNPs): rs12571751 in ZMIZ1, rs10842994 near KLHDC5, rs2796441 near TLE1, rs459193 near ANKRD55, rs10401969 in CILP2, rs12970134 near MC4R, rs7202877 near BCAR1, rs11063069 near CCND2, rs8108269 near GIPR, and rs8090011 in LAMA1 using a multiplex polymerase chain reaction invader assay. The association of each SNP locus with the disease was evaluated using a logistic regression analysis.ResultsAll SNPs examined in this study had the same direction of effect (odds ratio > 1.0, p = 9.77 × 10^-4, binomial test), as in the original reports. Among them, rs12571751 in ZMIZ1 was significantly associated with type 2 diabetes [p = 0.0041, odds ratio = 1.123, 95% confidence interval 1.037–1.215, adjusted for sex, age and body mass index (BMI)], but we did not observe significant association of the remaining 9 SNP loci with type 2 diabetes in the present Japanese population (p ≥ 0.005). A genetic risk score, constructed from the sum of risk alleles for the 7 SNP loci identified by un-stratified analyses in the European GWAS meta-analysis were associated with type 2 diabetes in the present Japanese population (p = 2.3 × 10^-4, adjusted for sex, age and BMI).ConclusionsZMIZ1 locus has a significant effect on conferring susceptibility to type 2 diabetes also in the Japanese population.     

赤霞珠葡萄果实苹果酸生物合成关键转录因子的筛选与鉴定

该研究以宁夏贺兰山东麓酿酒葡萄种植区栽培面积最大的‘赤霞珠’为材料,在前期完成从果实形成至成熟不同发育时期的转录组测序以及关键有机酸含量测定基础上,进一步通过转录因子结合位点预测、差异表达基因分析、加权基因共表达网络关联分析(WGCNA),逐步筛选出与‘赤霞珠’果实苹果酸生物合成相关功能基因特异结合的、影响苹果酸生物合成的相应转录因子,并对其进行qRT PCR验证,以揭示这些关键功能基因及其关键转录因子在葡萄不同种植区、果实不同发育时期存在的相互调控作用机制,为以后培育优质酿酒葡萄提供新的理论依据与思路。结果表明：(1)GC/MS分析发现, ‘赤霞珠’果实在4个发育时期的延胡索酸和苹果酸含量变化趋势基本一致,两种酸含量均从果实硬果期到绿果期逐步升至最高(3.63和626.53 μg/g),之后缓慢下降,经转色期到成熟期后逐渐降至最低(2.14和244.26 μg/g),而草酰乙酸的变化趋势却相反,在硬果期含量最高(315.54 μg/g),经绿果期、转色期到成熟期逐渐降至最低值(126.11 μg/g)。(2)‘赤霞珠’果实发育时期样本转录组测序共获得可能与苹果酸生物合成途径12种功能基因结合的转录因子6 411个,其中延胡索酸水化酶(FH)的3个功能基因有86个转录因子,苹果酸脱氢酶(MDH)的10个功能基因有717个转录因子。(3)转录组测序数据及其与有机酸含量WGCNA关联结果的Veen分析确定了‘赤霞珠’果实成熟过程中与苹果酸生物合成相关度最高的3个FH基因(VIT_14s0060g01700、VIT_13s0019g03330、VIT_07s0005g00880)、2个MDH基因(VIT_10s0003g01000、VIT_13s0019g05250)及相应的18个关键转录因子。(4)qRT PCR验证及相关性分析表明, FH基因VIT_13s0019g03330与其转录因子VIT_01s0011g06200、VIT_08s0056g01230以及MDH基因VIT_13s0019g05250与其转录因子VIT_06s0004g04960、VIT_10s0003g02070的表达水平与苹果酸的积累存在显著正相关关系,推测这4个关键转录因子可能通过调控功能基因的转录,综合影响‘赤霞珠’果实苹果酸的生物合成。